The broad long-term objective of our research is to reduce the incidence of ventilator-induced lung injury (VILI), characterized by pneumothorax, progressive impairment in pulmonary mechanics, alveolar cell dysfunction, and profound changes in lung fluid balance and blood-gas barrier permeability. In the previous funding periods we identified cyclic and tonic deformation magnitudes above which we measured acute alterations in epithelial cell viability and barrier properties. Moreover, the injurious deformation magnitudes we applied in vitro correlated well with tidal volumes and positive end-expiratory pressures used clinically that have been associated with clinical morbidity in ventilator induced lung injury (VILI). These findings underscore the potential relevance of using experimental models with carefully controlled conditions to identify the mechanisms responsible for stretch-induced disruption of the alveolar epithelial barrier, and to identify opportunities for injury intervention. The in vitro model for VILI that we established during the first 6 years of funding is limited to healthy rat alveolar epithelial cell monolayers exposed to 1 hr periods of deformation. In this competitive renewal our major objective is to determine specific stretch-induced mechanical and molecular signals that modulate alveolar epithelial permeability during clinically relevant conditions - including chronic continuous cycling and ARDS. In Aim 1 we hypothesize that the environmental stressors of sepsis, high cycling rates and long stretch durations each compromise epithelial monolayer barrier properties in both primary cell monolayers and intact lungs. In Aim 2 we hypothesize that stretch induces barrier alterations by activation of mitogen- activated protein kinases, which initiate nuclear factor kappa B dependent cytokine expression and release to ultimately increase monolayer permeability. In Aim 3 we will test the hypotheses that acute (<1 hr) stretch reorganizes the cytoskeleton, which increases monolayer permeability directly by dissociating tight junctional (TJ) proteins from actin, and indirectly by initiating kinase-mediated TJ phosphorylation and TJ complex disassembly. Thus, we build on the foundation established during previous funding periods - both to deepen our understanding of basic mechanisms and to enhance the clinical translation of our findings - and progress towards our goal of reducing acute VILI due to epithelial over-distension.